Compositions and methods for topical delivery of oligonucleotides

ABSTRACT

The present invention relates to compositions and methods which enhance the delivery of oligonucleotides and other nucleosidic moieties via topical routes of administration. Preferred compositions include liposomes or penetration enhancers for the delivery of such moieties to dermal and/or epidermal tissue in an animal for investigative, therapeutic or prophylactic purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/876,962, filed on Jun. 25, 2004, which application is a continuationof U.S. patent application Ser. No. 09/315294, filed on May 20, 1999,now U.S. Pat. No. 6,841,539, which is a continuation-in-part of U.S.patent application Ser. No. 09/082,336 filed on May 21, 1998, nowabandoned, the disclosure of each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to new compositions and methods for thetopical delivery of nucleic acids to the epidermis, the dermis, andstrata therein, of animals. More particularly, the present invention isdirected to the use of liposomes and penetration enhancers to effecttransport of oligonucleotides and other nucleic acids into the epidermisand dermis. More specific objectives and advantages of the inventionwill hereinafter be made clear or become apparent to those skilled inthe art during the course of explanation of preferred embodiments of theinvention.

BACKGROUND OF THE INVENTION

Due to recent advances in biotechnology, particularly in the field ofmolecular biology, there has been significant progress in the treatmentof diseases previously intractable, including cancers, genetic diseases,autoimmune disorders and AIDS. Many of these advances are achievedthrough the administration of nucleic acid molecules to a subject, oftenhuman. Often the administered nucleic acids are oligonucleotides.

The present invention is particularly drawn to compositions and methodsfor modulating the production of selected proteins or other biologicaleffectors in an animal, which involves the administration of nucleicacids, including oligonucleotides such as, for example, antisenseoligonucleotides, to the dermis and epidermis of an animal.

Various modes of administration of oligonucleotides to subjects havebeen shown to be effective for delivery of oligonucleotides toparticular tissues or organs for the treatment of several diseasesand/or disorders. For example, U.S. Pat. No. 5,595,978, issued Jan. 21,1997, to Draper et al., discloses intravitreal injection as a means forthe direct delivery of antisense oligonucleotides to the vitreous humorof the mammalian eye for the purpose of treating viral infectionsthereof. To date, however, attempts to effectively deliveroligonucleotides to the dermis and epidermis have not been realized.

The topical administration of oligonucleotides offers the promise ofsimpler, easier, and more effective delivery of nucleic acids to theepidermis and dermis without the need for sterile procedures and theirconcomitant expenses (e.g., hospitalization, physician fees, etc.).Thus, there is a need to provide compositions and methods for thetopical delivery of oligonucleotides to the epidermis and dermis, and toselected strata therein, of an animal. It is desirable that such novelcompositions and methods provide for the simple, efficient andconvenient delivery of therapeutic nucleic acids, especiallyoligonucleotides.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided for topical delivery of nucleic acids in an animal. Inparticular, the present invention provides compositions and methods formodulating the production of selected proteins or other biologicaleffectors in an animal, which involves the administration of anoligonucleotide, especially an antisense oligonucleotide, via topicalmeans to an animal, thereby circumventing the complications and expensewhich may be associated with intravenous and other parenteral modes ofin vivo administration.

“Topical administration” refers to the delivery of a nucleic acid to ananimal by contacting, directly or otherwise, a formulation comprisingthe oligonucleotide to all or a portion of the skin (epidermis) of ananimal. The term encompasses several routes of administration including,but not limited to, topical and transdermal. A common requirement forthese modes of administration is penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. In oneaspect, topical administration is used as a means to penetrate theepidermis and dermis and ultimately achieve systemic delivery ofoligonucleotides. In another aspect, topical administration is used as ameans to selectively deliver oligonucleotides to the epidermis or dermisof an animal, or to specific strata thereof.

Compositions of the present invention may be a mixture of components orphases as are present in emulsions (including microemulsions andcreams), and related formulations comprising two or more phases. In oneaspect, the pharmaceutical compositions of the invention comprise aplurality of at least one type of nucleic acid and a plurality of atleast one type of liposome. In certain embodiments, the nucleic acid isencapsulated, i.e., contained within the liposomes, while in others thenucleic acid is mixed with preformed liposomes to achieve anuncharacterized, presumably external, configuration with the liposomes;certain compositions of the invention comprise both types of[liposome:nucleic acid] configurations.

As detailed infra, the nucleic acid formulated in the pharmaceuticalcompositions of the invention can be, for example, an antisenseoligonucleotide, a ribozyme, a peptide nucleic acid (PNA), an externalguide sequence (EGS), a molecular decoy or an aptamer. The liposomeportion of the pharmaceutical compositions of the invention can be aneutral liposome, an anionic liposome, or a anionic fusogenic liposome.Preferred liposomes are formed from one or more phospholipids, such asdimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine.Particularly preferred liposomes are formed from a phospholipid,phosphatidylcholine derived from some natural source or a syntheticphosphatidylcholine molecule (hereinafter referred to in general as“phosphatidylcholine”), and a sterol such as, e.g., cholesterol. Ingeneral, the liposome is present in an amount which is effective todeliver the nucleosidic moiety to dermal or epidermal tissue in ananimal.

In another aspect, the pharmaceutical compositions of the inventioncomprise at least one nucleosidic moiety and at least one penetrationenhancer for enhancing penetration of the nucleosidic moiety into dermalor epidermal tissue in an animal. Representative penetration enhancersinclude fatty acids (such as isopropyl myristate), bile salts, chelatingagents, surfactants, and non-surfactants (such as unsaturated cyclicureas, 1-alkyl-alkanones, 1-alkenylazacyclo-alakanones, and steroidalanti-inflammatory agents), glycols, pyrrols, 1-acylazacycloheptan-2-ones(“azones”), and terpenes.

Also provided are methods for delivering nucleosidic moieties to dermalor epidermal tissue in an animal comprising one of the applying apharmaceutical composition of the invention to epidermal tissue. Incertain methods, the nucleosidic moiety is delivered preferentially tocells of the dermal tissue, while in other methods the nucleosidicmoiety is delivered preferentially to cells of the epidermal tissue.

Because of the advantages of topical delivery of drugs of the antisenseclass, the compositions and methods of the invention can be used intherapeutic methods as explained in more detail herein. The compositionsand methods herein provided may also be used to examine the function ofvarious proteins and genes in vitro in cultured or preserved dermaltissues and in animals. The invention can be thus applied to examine thefunction of any gene, including, in animal other than a human, thoseessential to animal development. The methods of the invention can alsobe used therapeutically or prophylactically, for example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

BRIEF DESCRIPTION OF THE FIGURES

The numerous objects and advantages of the present invention can bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIGS. 1 and 2 show in tabular form results of epidermal and dermaldelivery of oligonucleotides with various vehicle enhancers.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The invention is drawn to the topical administration of a nucleic acid,such as an oligonucleotide, having biological activity in an animal. By“having biological activity,” it is meant that the nucleic acidfunctions to modulate the expression of one or more genes in an animalas reflected in either absolute function of the gene (such as ribozymeactivity) or by production of proteins coded by such genes. In thecontext of this invention, “to modulate” means to either effect anincrease (stimulate) or a decrease (inhibit) in the expression of agene. Such modulation can be achieved by, for example, an antisenseoligonucleotide by a variety of mechanisms known in the art, includingbut not limited to transcriptional arrest; effects on RNA processing(capping, polyadenylation and splicing) and transportation; enhancementor reduction of cellular degradation of the target nucleic acid; andtranslational arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996,6:1).

The present invention provides methods and compositions for delivery ofnucleic acids, particularly oligonucleotides, to the epidermis and/ordermis of an animal to increase the bioavailability of the nucleic acidtherein. As used herein, the term “bioavailability” refers to the amountof the administered drug therapy (in this case the oligonucleotide) thatreaches and acts upon its target. The term is used for drugs whoseefficacy is measured relative to the concentration in the blood eventhough the ultimate site of action of the drug might be outside theblood, e.g., intracellular (see van Berge-Henegouwen et al.,Gastroenterology, 1977, 73, 300).

The compositions and methods of the invention may be used to provideprophylactic, palliative or therapeutic relief from a disease ordisorder that is treatable in whole or in part with one or more nucleicacids. In a preferred embodiment, such a disease or disorder istreatable in whole or in part via topical administration of an antisenseoligonucleotide to an animal having such a disease or disorder.

As used in the present invention, unless specified otherwise, the term“animal” refers to mammals including but not limited to humans andprimates; avians including chickens and turkeys; domestic household,sport or farm animals including dogs, cats, sheep, goats, cows, horsesand pigs; lab animals including rats, mice, rabbits and guinea pigs;fish; reptiles; and zoo animals.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum corneum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations which provide seals to further enhance theskin's permeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used. Consequently, thereis a need for compositions and methods to facilitate the transport ofnucleic acids through the skin's permeability barrier to the epidermisand the dermis.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

A preferred method for the delivery of biologically active substances tothe skin is topical administration. Topical administration can be usedas the route of administration when local delivery of a drug is desiredat, or immediately adjacent to, the point of application of the drugcomposition or formulation. Three general types of topical routes ofadministration include administration of a drug composition to mucousmembranes, skin or eyes.

Transdermal drug delivery is a valuable route for the administration oflipid soluble therapeutics. The dermis is more permeable than theepidermis and therefore absorption is much more rapid through abraded,burned or denuded skin. Inflammation and other physiologic conditionsthat increase blood flow to the skin also enhance transdermaladsorption. Absorption via this route may be enhanced by the use of anoily vehicle (injunction) or through the use of one or more penetrationenhancers. Other effective ways to deliver drugs via the transdermalroute include hydration of the skin and the use of controlled releasetopical patches. The transdermal route provides a potentially effectivemeans to deliver a drug for systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose deposition and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of drugs across mucosal sites in accordance with compositionsand methods of the present invention.

II. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The know-how on the preparation of suchcompositions and formulations is generally known to those skilled in thepharmaceutical and formulation arts and may be applied to theformulation of the compositions of the present invention.

A. Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 um indiameter. (Idson, in “Pharmaceutical Dosage Forms,” Lieberman, Riegerand Banker (Eds.), 1988, volume 1, p. 199; Rosoff, in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245; Block in “Pharmaceutical Dosage Forms,” Lieberman, Rieger andBanker (Eds.), 1988, volume 2, p. 335; Higuchi et al., in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water in oil (w/o) or of the oil inwater (o/w) variety. When an aqueous phase is finely divided into anddispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water in oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called an oil inwater (o/w) emulsion. Emulsions may contain additional components inaddition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil in water in oil (o/w/o) and water in oil in water (w/o/w) emulsions.

Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger andBanker (Eds.), 1988, volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 285; Idson, in“Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.),1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group into: nonionic, anionic, cationicand amphoteric (Rieger, in “Pharmaceutical Dosage Forms,” Lieberman,Rieger and Banker (Eds.), 1988, volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker(Eds.), 1988, volume 1, p. 335; Idson, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylc cellulose andcarboxypropyl cellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methylparaben, propylparaben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, boricacid and phenoxyethanol. Antioxidants are also commonly added toemulsion formulations to prevent deterioration of the formulation.Antioxidants used may be free radical scavengers such as tocopherols,alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

Preservatives used in any oligonucleotide formulation will preferablyhave a broad spectrum of antimicrobial activity and be compatible withhighly negatively charged oligonucleotides at neutral pH. To determinepreferred preservatives, oligonucleotides were incubated with variouspreservatives in the presence and absence of selected organisms[Staphylococcus aureus (ATCC No. 6538), Escherichia coli (ATCC No.8739), Candida albicans (ATCC No. 10231) and Aspergillus niger (ATCC No.16404)] according to USP 23 Antimicrobial Effectiveness Test (AET)procedures. According to results of these studies it has been discoveredthat preferred preservatives for oligonucleotide formulations include acombination of methylparaben, propylparaben and phenoxyethanol. Thetotal amount of the preservative combination will depend on the dosageform used but will in general be from about 0.1% to 20% by weight. Intopical emulsion compositions of the invention, the preservativecombination will be present in an amount from about 0.1% to 10%,preferably 0.5% to 8% and more preferably 1% to 5%. In a preferredembodiment, methylparaben and propylparaben will each be present in anamount from about 0.1% to 1% and phenoxyethanol in an amount from about1 to 5%. In a particularly preferred embodiment methylparaben,propylparaben and phenoxyethanol will be present in a ratio of about1:1:5 respectively.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in “Pharmaceutical Dosage Forms,” Lieberman,Rieger and Banker (Eds.), 1988, volume 1, p. 199). Emulsion formulationsfor oral delivery have been very widely used because of reasons of easeof formulation, efficacy from an absorption and bioavailabilitystandpoint. (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Riegerand Banker (Eds.), 1988, volume 1, p. 245; Idson, in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.199). Mineral-oil base laxatives, oil-soluble vitamins and high fatnutritive preparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman,Rieger and Banker (Eds.), 1988, volume 1, p. 545). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, in“Pharmaceutical Dosage Forms, ” Lieberman, Rieger and Banker (Eds.),1988, volume 1, p. 245; Block, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 335). Comparedto conventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO0750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

In a particularly preferred embodiment, emulsion compositions compriseisopropyl myristate (IPM) as an emollient. IPM emulsions of theinvention may be in cream form and incorporate IPM in an amount fromabout 1% to 50% by weight, more preferably 5% to 20% and most preferablyabout 10%. In preferred cream emulsions, glycerol monostearate serves asthe oil phase emulsifier while polyoxyl 40 stearate serves as the waterphase emulsifier, each present in an amount from about 1% to 30% andmore preferably 5% to 20%. In a particularly preferred embodiment,glycerol monostearate is present in an amount of about 10% by weight andpolyoxyl 40 stearate in an amount of about 15%. Preferred creamemulsions may further comprise viscosity-increasing agents such ashydroxypropyl methylcellulose. In a particularly preferred embodiment,hydroxypropyl methylcellulose is present in an amount from about 0.01%to about 5%, more preferably from 0.1% to 2% and most preferably about0.5%.

B. Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently-withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly transformable and able topass through such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell's cytoplasm (Wanget al., Biochem. Biophys. Res. Commun., 147 (1987) 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, Vol. 2, 405-410). Further, an additional study tested the efficacyof interferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 18, 1992, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome” I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome” II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes.

The-most common way of classifying and ranking the properties of themany different types of surfactants, both natural and synthetic, is bythe use of the hydrophile/lipophile balance (HLB). The nature of thehydrophilic group (also known as the ‘head’) provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., NewYork, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

C. Penetration Enhancers

In one embodiment, the present invention employ's various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Crit. Rev. Ther.Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classesof penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Crit. Rev. Ther. Drug Carrier Systems, 1991, p. 92); andperfluorhemical emulsions, such as FC-43 Takahashi et al., J. Pharm.Pharmacol., 1988, 40:252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Crit. Rev. Ther. Drug Carrier Systems, 1991, p. 92; Muranishi, Crit.Rev. Ther. Drug Carrier Systems, 1990, 7:1; El Hariri et al., J. Pharm.Pharmacol., 1992, 44:651).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pages 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7:1; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263:25;Yamashita et al., J. Pharm. Sci., 1990, 79:579).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7:1; Buur et al., J. Control Rel., 1990, 14:43).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7:1). This class ofpenetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39:621).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

D. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al.,Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).

E. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

F. Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecomposition of present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, hydroxypropyl methylcellulose,sodium carboxymethylcellulose, sorbitol and/or dextran. The suspensionmay also contain stabilizers.

III. Oligonucleotides

The present invention employs pharmaceutical compositions comprisingbiologically active oligonucleotides useful for prophylactic, palliativeor therapeutic purposes and, in isolated tissues or organs or in ananimal other than a human, for investigative use. Typically, theformulations of the invention will comprise an oligonucleotide in anamount of from about 0.005 ng/mL to about 400 mg/mL, preferably fromabout 0.01 ng/mL to about 200 mg/mL, most preferably from about 0.1ng/mL to about 100 mg/mL, where “about” indicates ±5% of the indicatedconcentration.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentintersugar (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

A discussion of antisense oligonucleotides and some desirablemodifications can be found in De Mesmaeker et al. (Acc. Chem. Res.,1995, 28, 366). Generally, oligonucleotides formulated in thecompositions of the invention may be from about 8 to about 100nucleotides in length, more preferably from about 10 to about 50nucleotides in length, and most preferably from about 10 about 25nucleotides in length.

Oligonucleotides that are formulated in the compositions of theinvention include (1) antisense compounds and (2) other bioactiveoligonucleotides. These compounds are described in more detail, infra.

A. Antisense Compounds: As used herein, the term “antisense compound”encompasses, inter alia, antisense oligonucleotides, antisense peptidenucleic acids (PNAs), ribozymes and EGSs (described infra). In antisensemodulation of messenger RNA (mRNA), hybridization of an antisensecompound with its mRNA target interferes with the normal role of mRNAand causes a modulation of its function in cells. The functions of mRNAto be interfered with include all vital functions such as translocationof the RNA to the site for protein translation, actual translation ofprotein from the RNA, splicing of the RNA to yield one or more mRNAspecies, turnover or degradation of the mRNA and possibly evenindependent catalytic activity which may be engaged in by the RNA. Theoverall effect of such interference with mRNA function is modulation ofthe expression of a protein, wherein “modulation” means either anincrease (stimulation) or a decrease (inhibition) in the expression ofthe protein. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression.

Antisense compounds can exert their effect by a variety of means. Onesuch means is the antisense-mediated direction of an endogenousnuclease, such as RNase H in eukaryotes or RNase P in prokaryotes, tothe target nucleic acid (Chiang et al., J. Biol. Chem., 1991, 266,18162; Forster et al., Science, 1990, 249, 783). The sequences thatrecruit RNase P are known as External Guide Sequences, hence theabbreviation “EGS” (Guerrier-Takada et al., Proc. Natl. Acad. Sci. USA,1997, 94, 8468). Another means involves covalently linking a syntheticmoiety having nuclease activity to an oligonucleotide having anantisense sequence, rather than relying upon recruitment of anendogenous nuclease. Synthetic moieties having nuclease activityinclude, but are not limited to, enzymatic RNAs, lanthanide ioncomplexes, and the like (Haseloff et al., Nature, 1988, 334, 585; Bakeret al., J. Am. Chem. Soc., 1997, 119, 8749).

As used herein, the term “antisense compound” also includes ribozymes,synthetic RNA molecules and derivatives thereof that catalyze highlyspecific endoribonuclease reactions (see, generally, U.S. Pat. No.5,543,508 to Haseloff et al. and U.S. Pat. No. 5,545,729 to Goodchild etal.). The cleavage reactions are catalyzed by the RNA moleculesthemselves. In naturally occurring RNA molecules, the sites ofself-catalyzed cleavage are located within highly conserved regions ofRNA secondary structure (Buzayan et al., Proc. Natl. Acad. Sci. USA,1986, 83, 8859; Forster et al., Cell, 1987, 50, 9). Naturally occurringautocatalytic RNA molecules have been modified to generate ribozymeswhich can be targeted to a particular cellular or pathogenic RNAmolecule with a high degree of specificity. Thus, ribozymes serve thesame general purpose as antisense oligonucleotides (i.e., modulation ofexpression of a specific gene) and, like oligonucleotides, are nucleicacids possessing significant portions of single-strandedness. That is,ribozymes have substantial chemical and functional identity with otherbioactive compounds and may thus be formulated for pharmaceuticaldelivery using the liposomes of the present invention.

The antisense compounds formulated in the compositions of the invention(1) may be from about 8 to about 100 nucleotides in length, morepreferably from about 10 to about 30 nucleotides in length, (2) aretargeted to a nucleic acid sequence required for the expression of agene from a mammal, including a human, and (3), when contacted withcells expressing the target gene, modulate its expression. Due to thebiological activity of the gene product encoded by the target gene,modulation of its expression has the desirable result of providingspecific prophylactic, palliative and/or therapeutic effects.

B. Other Bioactive Oligonucleotides: The term “Other BioactiveOligonucleotide” encompasses, inter alia, aptamers and molecular decoys(described infra). As used herein, the term is meant to refer to anyoligonucleotide (including a PNA) that (1) provides a prophylactic,palliative or therapeutic effect to an animal in need thereof and (2)acts by a non-antisense mechanism, i.e., by some means other than byhybridizing to a nucleic acid.

The name aptamer has been coined by Ellington et al. (Nature, 1990, 346,818) to refer to nucleic acid molecules that fit and therefore bind withsignificant specificity to non-nucleic acid ligands such as peptides,proteins and small molecules such as drugs and dyes. Because of thesespecific ligand binding properties, nucleic acids and oligonucleotidesthat may be classified as aptamers may be readily purified or isolatedvia affinity chromatography using columns that bear immobilized ligand.Aptamers may be nucleic acids that are relatively short to those thatare as large as a few hundred nucleotides. For example, RNA aptamersthat are 155 nucleotides long and that bind dyes such as Cibacron Blueand Reactive Blue 4 with good selectivity have been reported (Ellingtonet al., Nature, 1990, 346, 818). While RNA molecules were first referredto as aptamers, the term as used in the present invention refers to anynucleic acid or oligonucleotide that exhibits specific binding to smallmolecule ligands including, but not limited to, DNA, RNA, DNAderivatives and conjugates, RNA derivatives and conjugates, modifiedoligonucleotides, chimeric oligonucleotides, and gapmers (see, e.g.,U.S. Pat. No. 5,523,389, to Ecker et al., issued Jun. 4, 1996 andincorporated herein by reference).

Molecular decoys are short double-stranded nucleic acids (includingsingle-stranded nucleic acids designed to “fold back” on themselves)that mimic a site on a nucleic acid to which a factor, such as aprotein, binds. Such decoys are expected to competitively inhibit thefactor; that is, because the factor molecules are bound to an excess ofthe decoy, the concentration of factor bound to the cellular sitecorresponding to the decoy decreases, with resulting therapeutic,palliative or prophylactic effects. Methods of identifying andconstructing decoy molecules are described in, e.g., U.S. Pat. No.5,716,780 to Edwards et al.

Another type of bioactive oligonucleotide is an RNA-DNA hybrid moleculethat can direct gene conversion of an endogenous nucleic acid(Cole-Strauss et al., Science, 1996, 273, 1386). Any of the precedingbioactive oligonucleotides may be formulated in the liposomes of theinvention and used for prophylactic or therapeutic purposes.

C. Oligonucleotide Modifications

An oligonucleotide is a polymer of a repeating unit generically known asa nucleotide. An unmodified (naturally occurring) nucleotide has threecomponents: (1) a nitrogen-containing heterocyclic base linked by one ofits nitrogen atoms to (2) a 5-pentofuranosyl sugar and (3) a phosphateesterified to one of the 5′ or 3′ carbon atoms of the sugar. Whenincorporated into an oligonucleotide chain, the phosphate of a firstnucleotide is also esterified to an adjacent sugar of a second, adjacentnucleotide via a 3′-5′ phosphate linkage.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be further joined to form a circular structure, however,within the context of the invention, open linear structures aregenerally preferred.

Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the intersugar “backbone” of the oligonucleotide.The normal linkage or backbone of RNA and DNA is a 3′ to 5′phosphodiester linkage. The backbone of an oligonucleotide (or otherantisense compound) positions a series of bases in a specific order; thewritten representation of this ordered series of bases, usually writtenin 5′ to 3′ order unless otherwise indicated, is known as a nucleotideor nucleobase sequence.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” In the context of the invention, “hybridization” meanshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother.

“Specifically hybridizable” and “complementary” are thus terms which areused to indicate a sufficient degree of complementarity or precisepairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. An oligonucleotide isspecifically hybridizable to its target sequence due to the formation ofbase pairs between specific partner nucleobases in the interior of anucleic acid duplex. Among the naturally occurring nucleobases, guanine(G) binds to cytosine (C), and adenine (A) binds to thymine (T) oruracil (U). In addition to the equivalency of U (RNA) and T (DNA) aspartners for A, other naturally occurring nucleobase equivalents areknown, including 5-methylcytosine and 5-hydroxymethylcytosine (HMC) (Cequivalents), and 5-hydroxymethyluracil (U equivalent). Furthermore,synthetic nucleobases which retain partner specificity are known in theart and include, for example, 7-deaza-Guanine, which retains partnerspecificity for C. Thus, an oligonucleotide's capacity to specificallyhybridize with its target sequence will not be altered by a chemicalmodification to a nucleobase in the nucleotide sequence of theoligonucleotide which does not impact its specificity for a partnernucleobase in the target nucleic acid.

It is understood in the art that the nucleobase sequence of anoligonucleotide or other antisense compound need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An antisense compound is specifically hybridizable to itstarget nucleic acid when there is a sufficient degree of complementarityto avoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, or, in the case of in vitro assays, under assayconditions.

Antisense oligonucleotides are commonly used as research reagents,diagnostic aids, and therapeutic agents.

For example, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes, forexample to distinguish between the functions of various members of abiological pathway. This specific inhibitory effect has, therefore, beenharnessed by those skilled in the art for research uses. The specificityand sensitivity of oligonucleotides is also harnessed by those of skillin the art for therapeutic uses. Specific examples of preferredantisense compounds useful in this invention include oligonucleotidescontaining modified backbones or non-natural intersugar linkages. Asdefined in this specification, oligonucleotides having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described in thefollowing subsections. It is not necessary for all positions in a givencompound to be uniformly modified, and in fact more than one of thefollowing modifications may be incorporated in a single antisensecompound or even in a single residue thereof, for example, at a singlenucleoside within an oligonucleotide.

Modified Linkages: Preferred modified oligonucleotide backbones include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalklyphosphotriesters, and boranbphosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

Representative United States Patents that teach the preparation of theabove phosphorus atom containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 255,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein (i.e., oligonucleosides) have backbones that areformed by short chain alkyl or cycloalkyl intersugar linkages, mixedheteroatom and alkyl or cycloalkyl intersugar linkages, or one or moreshort chain heteroatomic or heterocyclic intersugar linkages. Theseinclude those having morpholino linkages (formed in part from the sugarportion of a nucleoside); siloxane backbones; sulfide, sulfoxide andsulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene containing backbones;sulfamate backbones; methyleneimino and methylenehydrazino backbones;sulfonate and sulfonamide backbones; amide backbones; and others havingmixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theintersugar linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al. (Science, 1991, 254, 1497).

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [(wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Nucleobases: The compounds of the invention may additionally oralternatively comprise nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Id., pages276-278) and are presently preferred base substitutions, even moreparticularly when combined with 2′-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/762,488, filed onDec. 10, 1996, also herein incorporated by reference.

Sugar Modifications: The antisense compounds of the invention mayadditionally or alternatively comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl, O—, S—, or N-alkenyl, or O,S— or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, also known as2′-O—(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995,78, 486), i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, as described in co-owned U.S. patent application Ser.No. 09/016,520, filed on Jan. 30, 1998, the contents of which are hereinincorporated by reference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/468,037, filed onJun. 5, 1995, also herein incorporated by reference.

Other Modifications: Additional modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminalnucleotide. For example, one additional modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327;Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but-are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned, and each of which is herein incorporated byreference.

Chimeric Oligonucleotides: The present invention also includes antisensecompounds which are chimeric compounds. “Chimeric” antisense compoundsor “chimeras,” in the context of this invention, are antisensecompounds, particularly oligonucleotides, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioateoligodeoxynucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart. RNase H-mediated target cleavage is distinct from the use ofribozymes to cleave nucleic acids, and ribozymes are not comprehended bythe present invention.

By way of example, such “chimeras” may be “gapmers,” i.e.,oligonucleotides in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for, e.g., RNase H, and the 5′ and3′ portions (the “wings”) are modified in such a fashion so as to havegreater affinity for, or stability when duplexed with, the target RNAmolecule but are unable to support nuclease activity (e.g., 2′-fluoro-or 2′-methoxyethoxy-substituted). Other chimeras include “hemimers,”that is, oligonucleotides in which the 5′ portion of the oligonucleotideserves as a substrate for, e.g., RNase H, whereas the 3′ portion ismodified in such a fashion so as to have greater affinity for, orstability when duplexed with, the target RNA molecule but is unable tosupport nuclease activity (e.g., 2′-fluoro- or2′-methoxyethoxy-substituted), or vice-versa.

A number of chemical modifications to oligonucleotides that confergreater oligonucleotide:RNA duplex stability have been described byFreier et al. (Nucl. Acids Res., 1997, 25, 4429). Such modifications arepreferred for the RNase H-refractory portions of chimericoligonucleotides and may generally be used to enhance the affinity of anantisense compound for a target RNA.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned, and each of which is hereinincorporated by reference, and commonly owned and allowed U.S. patentapplication Ser. No. 08/465,880, filed on Jun. 6, 1995, also hereinincorporated by reference.

A further preferred modification includes 2′-dimethylamino oxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inco-owned U.S. patent application Ser. No. 09/016,520, filed on Jan. 30,1998, the contents of which are herein incorporated by reference. Otherpreferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the sugar group, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Thenucleosides of the oligonucleotides may also have sugar mimetics such ascyclobutyl moieties in place of the pentofuranosyl sugar.

Unsubstituted and substituted phosphodiester oligonucleotides arealternately synthesized on an automated DNA synthesizer (AppliedBiosystems model 380B) using standard phosphoramidite chemistry withoxidation by iodine.

Phosphorothioates are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step..After cleavage from the CPG column and deblocking in concentratedammonium hydroxide at 55° C. (18 hr), the oligonucleotides were purifiedby precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.Nos. 5,256,775 or 5,366,878, hereby incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Boranophosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand PO or PS linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5. They may also be prepared in accordance with U.S.Pat. Nos. 5,539,082; 5,700,922, and 5,719,262, herein incorporated byreference.

Examples of specific oligonucleotides and the target genes which theyinhibit, that may be employed in formulations of the present inventioninclude:

ISIS-2302 GCCCA AGCTG GCATC CGTCA (SEQ ID NO: 1) ICAM-1 ISIS-15839 GCCCAAGCTG GC AT C   C GT C A (SEQ ID NO: 1) ICAM-1 ISIS-1939 CCCCC ACCACTTCCC CTCTC (SEQ ID NO: 2) ICAM-1 ISIS-2503 TCCGT CATCG CTCCT CAGGG (SEQID NO: 4) Ha-ras ISIS-2922 GCGTT TGCTC TTCTT CTTGC G (SEQ ID NO: 5) HCMVISIS-13312 G C GTT TG CTC TTC TT C TTG C  G (SEQ ID NO: 5) HCMVISIS-3521 GTTCT CGCTG GTGAG TTTCA (SEQ ID NO: 6) PKCα ISIS-9605 GTT C TC GCTG GTGAG TTT C A (SEQ ID NO: 6) PKCα ISIS-9606 GTT C T C GCTG GTGAGTTT C A (SEQ ID NO: 6) PKCα ISIS-14859 AACTT GTG C T TG C T C (SEQ IDNO: 7) PKCα ISIS-5132 TCCCG CCTGT GACAT GCATT (SEQ ID NO: 8) c-rafISIS-14803 GTGCT CATGG TGCAC GGTCT (SEQ ID NO: 9) HCV ISIS-28089 GTGTGCCAGA CACCC TAT C T (SEQ ID NO: 10) TNFα ISIS-104838 G C TGA TTAGA GAGAGGT CCC (SEQ ID NO: 11) TNFα ISIS-2105 TTGCT TCCAT CTTCC TCGTC (SEQ IDNO: 12) HPVwherein (i) each oligo backbone linkage is a phosphorothioate linkage(except ISIS-9605) and (ii) each sugar is 2′-deoxy unless represented inbold font in which case it incorporates a 2′-O-methoxyethyl group andiii) underlined cytosine nucleosides incorporate a 5-methyl substituenton their nucleobase. ISIS-9605 incorporates natural phosphodiester bondsat the first five and last five linkages with the remainder beingphosphorothioate linkages.

D. Synthesis of Oligonucleotides: The oligonucleotides used inaccordance with this invention may be conveniently and routinely madethrough the well-known technique of solid phase synthesis. Equipment forsuch synthesis is sold by several vendors including, for example,Applied Biosystems (Foster City, Calif.). Any other means for suchsynthesis known in the art may additionally or alternatively beemployed. It is also known to use similar techniques to prepare otheroligonucleotides such as the phosphorothioates and alkylatedderivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents or pendingpatent applications, each of which is commonly assigned with thisapplication: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, issued Jun. 29, 1993, and U.S. Pat. No. 5,608,046, bothdrawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos.5,602,240, and 5,610,289, drawn to backbone modified oligonucleotideanalogs; and U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

E. Bioequivalents: In addition to oligonucleotide drugs per se, thepharmaceutical compositions of the present invention can be used toformulate any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other compound which, upon administration to ananimal including a human, is capable of providing (directly orindirectly) a biologically active oligonucleotide or residue thereof.Accordingly, for example, the disclosure is also drawn to “prodrugs” and“pharmaceutically acceptable salts” of the oligonucleotides of theinvention, pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents.

Oligonucleotide Prodrugs: The oligonucleotide and nucleic acid compoundsemployed in the compositions of the present invention may additionallyor alternatively be prepared to be delivered in a “prodrug” form. Theterm “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theantisense compounds may be prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 (Gosselin et al., published Dec. 9, 1993).

Pharmaceutically Acceptable Salts: The term “pharmaceutically acceptablesalts” refers to physiologically and pharmaceutically acceptable saltsof the oligonucleotide and nucleic acid compounds employed in thecompositions of the present invention (i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, ammonium, polyamines such as spermine and spermidine, and thelike. Examples of suitable amines are chloroprocaine, choline,N,N′-dibenzylethylenediamine, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66:1).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

n-1 Derivatives: During the process of oligonucleotide synthesis,nucleoside monomers are attached to the chain one at a time in arepeated series of chemical reactions such as nucleoside monomercoupling, oxidation, capping and detritylation. The stepwise yield foreach nucleoside addition is above 99%. That means that less than 1% ofthe sequence chain failed to be generated from the nucleoside monomeraddition in each step as the total results of the incomplete couplingfollowed by the incomplete capping, detritylation and oxidation (Smith,Anal. Chem., 1988, 60, 381A). All the shorter oligonucleotides, rangingfrom (n-1), (n-2), etc., to 1-mers (nucleotides), are present asimpurities in the n-mer olignucleotide product. Among the impurities,(n-2)-mer and shorter oligonucleotide impurities are present in verysmall amounts and can be easily removed by chromatographic purification(Warren et al., Chapter 9 In: Methods in Molecular Biology, Vol. 26:Protocols for Oligonucleotide Conjugates, Agrawal, S., Ed., 1994, HumanaPress Inc., Totowa, N.J., pages 233-264). However, due to the lack ofchromatographic selectivity and product yield, some (n-1)-mer impuritiesare still present in the full-length (i.e., n-mer) oligonucleotideproduct after the purification process. The (n-1) portion consists ofthe mixture of all possible single base deletion sequences relative tothe n-mer parent oligonucleotide. Such (n-1) impurities can beclassified as terminal deletion or internal deletion sequences,depending upon the position of the missing base (i.e., either at the 5′or 3′ terminus or internally). When an oligonucleotide containing singlebase deletion sequence impurities is used as a drug (Crooke, HematologicPathology, 1995, 9, 59), the terminal deletion sequence impurities willbind to the same target mRNA as the full length sequence but with aslightly lower affinity. Thus, to some extent, such impurities can beconsidered as part of the active drug component, and are thus consideredto be bioequivalents for purposes of the present invention.

IV. Therapeutic Indications and Other Uses

Psoriasis: One therapeutic indication of particular interest for topicaldelivery of oligonucleotides and other nucleic acids is psoriasis.Psoriasis is a common chronic and recurrent disease characterized bydry, well-circumscribed, silvery, scaling papules and plaques of varioussizes. The disease varies in severity from a few lesions to widespreaddermatosis with disabling arthritis or exfoliation. The ultimate causeof psoriasis is not known, but the thick scaling that occurs is probablydue to increased epidermal cell proliferation (The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 2283-2285, Berkow et al., eds.,Rahway, N.J., 1987). Inhibitors of Protein Kinase C (PKC), ICAM-1 andtumour necrosis factor (TNFα) have been shown to have bothantiproliferative and anti-inflammatory effects in vitro. Someantipsoriasis drugs, such as cyclosporin A and anthralin, have beenshown to inhibit PKC, and inhibition of PKC has been suggested as atherapeutic approach to the treatment of psoriasis (Hegemann, L. and G.Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp. 357-368, CRCPress, Boca Raton, Fla., 1992).

Antisense compounds targeted to Protein Kinase C (PKC) proteins aredescribed in U.S. Pat. No. 5,620,963 to Cook et al. and U.S. Pat. No.5,681,747 to Boggs et al.

Inflammations Another type of therapeutic indication of particularinterest for topical modes of delivery includes inflammatory disordersof the skin. These occur in a variety of forms including, for example,lichen planus, toxic epidermal necrolyis (TEN), ertythema multiforme andthe like (The Merck Manual of Diagnosis and Therapy, 15th Ed., pp.2286-2292, Berkow et al., eds., Rahway, N.J., 1987). Expression ofICAM-1 has been associated with a variety of inflammatory skin disorderssuch as allergic contact dermatitis, fixed drug eruption, lichen planusand psoriasis (Ho et al., J. Am. Acad. Dermatol., 1990, 22, 64;Griffiths et al., Am. J. Pathology, 1989, 135, 1045; Lisby et al., Br.J. Dermatol., 1989, 120, 479; Shiohara et al., Arch. Dermatol., 1989,125, 1371; Regezi et al., Oral Sure. Oral Med. Oral Pathol., 1996, 81,682). Moreover, intraperitoneal administration of a monoclonal antibodyto ICAM-1 decreases ovalbumin-induced eosinophil infiltration into skinin mice (Hakugawa et al., J. Dermatol., 1997, 24, 73). Antisensecompounds targeted to ICAM-1 are described in U.S. Pat. Nos. 5,514,788and 5,591,623, and co-pending U.S. patent application Ser. Nos.09/009,490 and 09/062,416, Jan. 20, 1998 and Apr. 17, 1998,respectively, all to Bennett et al.

Other antisense targets for skin inflammatory disorders are VCAM-1 andPECAM-1. Intraperitoneal administration of a monoclonal antibody toVCAM-1 decreases ovalbumin-induced eosinophil infiltration into the skinof mice (Hakugawa et al., J. Dermatol., 1997, 24, 73). Antisensecompounds targeted to VCAM-1 are described in U.S. Pat. Nos. 5,514,788and 5,591,623. PECAM-1 proteins are glycoproteins which are expressed onthe surfaces of a: variety of cell types (for reviews, see Newman, J.Clin. Invest., 1997, 99, 3 and DeLisser et al., Immunol. Today, 1994,15, 490). In addition to directly participating in cell-cellinteractions, PECAM-1 apparently also regulates the activity and/orexpression of other molecules involved in cellular interactions (Litwinet al., J. Cell Biol., 1997, 139, 219) and is thus a key mediator ofseveral cell:cell interactions. Antisense compounds targeted to PECAM-1are described in co-pending U.S. patent application Ser. No. 09/044,506,filed Mar. 19, 1998, by Bennett et al.

Skin Cancers: Another type of therapeutic indication of interest fortopical delivery of oligonucleotides encompasses a variety of cancers ofthe skin. Representative skin cancers include benign tumors (warts,moles and the like) and malignant tumors such as, for-example, basalcell carcinoma, squamous cell carcinoma, malignant melanoma, Paget'sdisease, Kaposi's sarcoma and the like (The Merci Manual of Diagnosisand Therapy, 15th Ed., pp. 2301-2310, Berkow et al., eds., Rahay, N.J.,1987). A number of molecular targets involved in tumorigenesis,maintenance of the hyperproliferative state and metastasis are targetedto prevent or inhibit skin cancers, or to prevent their spread to othertissues.

The ras oncogenes are guanine-binding proteins that have been implicatedin cancer by, e.g., the fact that activated ras oncogenes have beenfound in about 30% of human tumors generally; this figure approached100% in carcinomas of the exocrine pancreas (for a review, see Downward,Trends in Biol. Sci., 1990, 15, 469). Antisense compounds targeted toH-ras and K-ras are described in U.S. Pat. No. 5,582,972 to Lima et al.,U.S. Pat. No. 5,582,986 to Monia et al. and U.S. Pat. No. 5,661,134 toCook et al., and in published PCT application WO 94/08003.

Protein Kinase C (PKC) proteins have also been implicated intumorigenesis. Antisense compounds targeted to Protein Kinase C (PKC)proteins are described in U.S. Pat. No. 5,620,963 to Cook et al. andU.S. Pat. No. 5,681,747 to Boggs et al. Also of interest are AP-1subunits and JNK proteins, particularly in regard to their roles intumorigenesis and metastasis. The process of metastasis involves asequence of events wherein (1) a cancer cell detaches from itsextracellular matrices, (2) the detached cancer cell migrates to anotherportion of an animal's body, often via the circulatory system, and (3)attaches to a distal and inappropriate extracellular matrix, therebycreated a focus from which a secondary tumor can arise. Normal cells donot possess the ability to invade or metastasize and/or undergoapoptosis (programmed cell death) if such events occur (Ruoslahti, Sci.Amer., 1996, 275, 72). However, many human tumors have elevated levelsof activity of one or more matrix metalloproteinases (MMPs)(Stetler-Stevenson et al., Annu. Rev. Cell Biol., 1993, 9, 541; Bernhardet al., Proc. Natl. Acad. Sci. (U.S.A.), 1994, 91, 4293. The MMPs are afamily of enzymes which have the ability to degrade components of theextracellular matrix (Birkedal-Hansen, Current Op. Biol., 1995, 7, 728).In particular, one member of this family, matrix metalloproteinase-9(MMP-9), is often found to be expressed only in tumors and otherdiseased tissues (Himelstein et al., Invasion & Metastasis, 1994, 14,246).

Several studies have shown that regulation of the MMP-9 gene may becontrolled by the AP-1 transcription factor (Kerr et al., Science, 1988,242, 1242; Kerr et al., Cell, 1990, 61, 267; Gum et al., J. Biol. Chem.,1996, 271, 10672; Hua et al., Cancer Res., 1996, 56, 5279). Inhibitionof AP-1 function has been shown to attenuate MMP-9 expression (U.S.patent application Ser. No. 08/837,201). AP-1 is a heterodimeric proteinhaving two subunits, the gene products of fos and jun. Antisensecompounds targeted to c-fos and c-jun are described in co-pending U.S.patent application Ser. No. 08/837,201, filed Mar. 14, 1997, by Dean etal.

Furthermore, AP-1 is itself activated in certain circumstances byphosphorylation of the Jun subunit at an amino-terminal position by JunN-terminal kinases (JNKs). Thus, inhibition of one or more JNKs isexpected to result in decreased AP-1 activity and, consequentially,reduced MMP expression. Antisense compounds targeted to JNKs aredescribed in co-pending U.S. patent application Ser. No. 08/910,629,filed Aug. 13, 1997, by Dean et al.

Infectious Diseases of the Skins Also of interest for topicalformulations of oligonucleotides are infectious diseases of the skin.Such infections are caused by viral, bacterial or fungal agents.

In the case of Lyme disease, the tick borne causative agent thereof, thespirochete Borrelia burgdorferi, up-regulates the expression of ICAM-1,VCAM-1 and ELAM-1 on endothelial cells in vitro (Boggemeyer et al., CellAdhes. Comm., 1994, 2, 145). Furthermore, it has been proposed that themediation of the disease by the anti-inflammatory agent prednisolone isdue in part to mediation of this up-regulation of adhesion molecules(Hurtenbach et al., Int. J. Immunopharmac., 1996, 18, 281). Thus,potential targets for therapeutic mediation (or prevention) of Lymedisease include ICAM-1, VCAM-1 and ELAM-1 (supra).

Other infectious disease of the skin which are tractable to treatmentusing the compositions and methods of the invention include disordersresulting from infection by bacterial, viral or fungal agents (The MerckManual of Diagnosis and Therapy, 15th Ed., pp. 2263-2277, Berkow et al.,eds., Rahay, N.J., 1987). With regards to infections of the skin causedby fungal agents, U.S. Pat. No. 5,691,461 provides antisense compoundsfor inhibiting the growth of Candida albicans.

With regards to infections of the skin caused by viral agents, U.S. Pat.Nos. 5,166,195, 5,523,389 and 5,591,600 provide oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting its replication. U.S. Pat. Nos.5,194,428 and 5,580,767 provide antisense compounds having antiviralactivity against influenza virus. U.S. Pat. No. 4,806,463 providesantisense compounds and methods using them to inhibit HTLV-IIIreplication. U.S. Pat. Nos. 4,689,320, 5,442,049, 5,591,720 and5,607,923 are directed to antisense compounds as antiviral agentsspecific to cytomegalovirus (CMV). U.S. Pat. No. 5,242,906 providesantisense compounds useful in the treatment of latent Epstein-Barr virus(EBV) infections. U.S. Pat. Nos. 5,248,670, 5,514,577 and 5,658,891provide antisense compounds useful in the treatment of herpes virusinfections. U.S. Pat. Nos. 5,457,189 and 5,681,944 provide antisensecompounds useful in the treatment of papilloma virus infections. Theantisense compounds disclosed in these patents, which are hereinincorporated by reference, may be used with the compositions of theinvention to effect prophylactic, palliative or therapeutic relief fromdiseases caused or exacerbated by the indicated pathogenic agents.

Investigative Uses: Antisense oligonucleotides employed in thecompositions of the present invention may also be used to determine thenature, function and potential relationship of various geneticcomponents of the body to disease or body states in animals. Heretofore,the function of a gene has been chiefly examined by the construction ofloss-of-function mutations in the gene (i.e., “knock-out” mutations) inan animal (e.g., a transgenic mouse). Such tasks are difficult,time-consuming and cannot be accomplished for genes essential to animaldevelopment since the “knock-out” mutation would produce a lethalphenotype. Moreover, the loss-of-function phenotype cannot betransiently introduced during a particular part of the animal's lifecycle or disease state; the “knock-out” mutation is always present.“Antisense knockouts,” that is, the selective modulation of expressionof a gene by antisense oligonucleotides, rather than by direct geneticmanipulation, overcomes these limitations (see, for example, Albert etal., Trends in Pharmacological Sciences, 1994, 15, 250). In addition,some genes produce a variety of mRNA transcripts as a result ofprocesses such as alternative splicing; a “knock-out” mutation typicallyremoves all forms of mRNA transcripts produced from such genes and thuscannot be used to examine the biological role of a particular mRNAtranscript. Antisense oligonucleotides have been systemicallyadministered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-a, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al., Nature, 1993, 363:260; Dean et al., Proc. Natl.Acad. Sci. U.S.A., 1994, 91:11762; and Wahlestedt et al., Science, 1993,259:528, respectively). In instances where complex families of relatedproteins are being investigated, “antisense knockouts” (i.e., inhibitionof a gene by systemic administration of antisense oligonucleotides) mayrepresent the most accurate means for examining a specific member of thefamily (see, generally, Albert et al., Trends Pharmacol. Sci., 1994,15:250). By providing compositions and methods for the simplenon-parenteral delivery of oligonucleotides and other nucleic acids, thepresent invention overcomes these and other shortcomings.

V. Treatment Regimens

The administration of therapeutic or pharmaceutical compositionscomprising the liposomes of the invention is believed to be within theskill of those in the art. In general, a patient in need of therapy orprophylaxis is administered a composition comprising a liposomallyformulated bioactive agents in accordance with the invention, commonlyin a pharmaceutically acceptable carrier, in doses ranging from 0.01 ugto 100 g per kg of body weight depending on the age of the patient andthe severity of the disorder or disease state being treated. Dosing isdependent on severity and responsiveness of the disease state to betreated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution orprevention of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual antisensecompounds, and can generally be estimated based on EC₅₀s found to beeffective in in vitro and in vivo animal models.

In the context of the invention, the term “treatment regimen” is meantto encompass therapeutic, palliative and prophylactic modalities ofadministration of one or more liposomal compositions of the invention. Aparticular treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of the liposomalcomposition may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disorder or diseasestate is observed, or if the disorder or disease state has been ablated.

An optimal dosing schedule is used to deliver a therapeuticallyeffective amount of the bioactive agent encapsulated within theliposomes of the invention being administered via a particular mode ofadministration. The term “therapeutically effective amount,” for thepurposes of the invention, refers to the amount ofoligonucleotide-containing pharmaceutical composition which is effectiveto achieve an intended purpose without undesirable side effects (such astoxicity, irritation or allergic response). Although individual needsmay vary, determination of optimal ranges for effective amounts ofpharmaceutical compositions is within the skill of the art. Human dosescan be extrapolated from animal studies (Katocs et al., Chapter 27 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990). Generally, the dosage required toprovide an effective amount of a pharmaceutical composition, which canbe adjusted by one skilled in the art, will vary depending on the age,health, physical condition, weight, type and extent of the disease ordisorder of the recipient, frequency of treatment, the nature ofconcurrent therapy (if any) and the nature and scope of the desiredeffect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds.,McGraw-Hill, New York, N.Y., 1996).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the bioactive agent is administered in maintenance doses,ranging from 0.01 ug to 100 g per kg of body weight, once or more daily,to once every years. For example, in the case of in individual known orsuspected of being prone to an autoimmune or inflammatory condition,prophylactic effects may be achieved by administration of preventativedoses, ranging from 0.01 ug to 100 g per kg of body weight, once or moredaily, to once every 20 years. In like fashion, an individual may bemade less susceptible to an inflammatory condition that is expected tooccur as a result of some medical treatment, e.g., graft versus hostdisease resulting from the transplantation of cells, tissue or an organinto the individual.

Prophylactic modalities for high risk individuals are also encompassedby the invention. As used herein, the term “high risk individual” ismeant to refer to an individual for whom it has been determined, via,e.g., individual or family history or genetic testing, that there is asignificantly higher than normal probability of being susceptible to theonset or recurrence of a disease or disorder. For example, a subjectanimal could have a personal and/or family medical history that includesfrequent occurrences of a particular disease or disorder.

As another example, a subject animal could have had such asusceptibility determined by genetic screening according to techniquesknown in the art (see, e.g., U.S. Congress, Office of TechnologyAssessment, Chapter 5 In: Genetic Monitoring and Screening in theWorkplace, OTA-BA-455, U.S. Government Printing Office, Washington,D.C., 1990, pages 75-99). As part of a treatment regimen for a high riskindividual, the individual can be prophylactically treated to preventthe onset or recurrence of the disease or disorder. The term“prophylactically effective amount” is meant to refer to an amount of apharmaceutical composition which produces an effect observed as theprevention of the onset or recurrence of a disease or disorder.Prophylactically effective amounts of a pharmaceutical composition aretypically determined by the effect they have compared to the effectobserved when a second pharmaceutical composition lacking the activeagent is administered to a similarly situated individual.

From in vivo animal studies wherein oligonucleotides have beenadministered topically or intradermally it has been shown thatoligonucleotides become widely distrubuted from the site ofadministration. For example oligonucleotide ISIS-2302 was topicallyapplied on the back of mini pigs and rats. Samples of dermal andepidermal tissue analyzed by capillary gel electrophoresis andimmunohistochemical staining detected significant levels of theoligonucleotide not only at the administration site (back) but also onstomach, neck and hind leg. Accordingly there is provided a method fordelivering an oligonucleotide to a first dermal or epidermal tissue sitein an animal comprising applying said oligonucleotide to a second dermalor epidermal tissue site in said animal wherein said first site isremoved from said second site. In preferred embodiments, theoligonucleotide is administered topically in a pharmaceuticalcomposition of the invention, in particular in an emulsion as describedherein. The method is particularly useful for ensuring delivery ofoligonucleotide evenly to dermal or epidermal tissue and/or over a greatarea or to sites that would otherwise be difficult to apply or would besensitive-to direct administration.

EXAMPLES

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

Example 1 Oligonucleotides

A. General Synthetic Techniques: Oligonucleotides were synthesized on anautomated DNA synthesizer using standard phosphoramidite chemistry withoxidation using iodine. Beta-cyanoethyldiisopropyl phosphoramidites werepurchased from Applied Biosystems (Foster City, Calif.). Forphosphorothioate oligonucleotides, the standard oxidation bottle wasreplaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxidein acetonitrile for the stepwise thiation of the phosphite linkages.

B. Oligonucleotide Purification: After cleavage from the controlled poreglass (CPG) column (Applied Biosystems) and deblocking in concentratedammonium hydroxide, at 55° C. for 18 hours, the oligonucleotides werepurified by precipitation 2× from 0.5 M NaCl with 2.5 volumes of ethanolfollowed by further purification by reverse phase high liquid pressurechromatography (HPLC). Analytical gel electrophoresis was accomplishedin 20% acrylamide, 8 M urea and 45 mM Tris-borate buffer (pH 7).

C. Oligonucleotide Labeling: In order to follow the distribution ofoligonucleotides in situ were radiolabelled to high specific activity bysynthetic incorporation of 35S using hydrogen phosphonate chemistryessentially as described by Stein et al. (Anal. Biochem., 1990, 188,11).

D. Oligonucleotide Structure: The oligonucleotides used in the studiesdescribed herein have the following structures and biologicalactivities.

ISIS 2302 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-GCC-CAA-GCT-GGC-ATC-CGT-CA-3′ (SEQ IDNO:1). ISIS 2302 is targeted to the 3′-untranslated region (3′-UTR) ofthe human ICAM-1 gene. ISIS 2302 is described in U.S. Pat. Nos.5,514,788 and 5,591,623, hereby incorporated by reference.

ISIS 1939 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-CCC-CCA-CCA-CTT-CCC-CTC-TC-3′ (SEQ IDNO:2). ISIS 1939 is targeted to the 3′-untranslated region (3′-UTR) ofthe human ICAM-1 gene. ISIS 1939 is described in U.S. Pat. Nos.5,514,788 and 5,591,623, hereby incorporated by reference.

ISIS 15839 is a phosphorothioate isosequence “hemimer” derivative ofISIS 2302 having the structure 5′-GCC-CAA-GCT-GGC-ATC-CGT-CA-3′ (SEQ IDNO:1), wherein emboldened “C” residues have 5-methylcytosine (m5c) basesand wherein the emboldened, double-underlined residues further comprisea 2′-methoxyethoxy modification (other residues are 2′-deoxy). ISIS15839 is described in copending U.S. patent application Ser. No.09/062,416, filed Apr. 17, 1998, hereby incorporated by reference.

ISIS 3082 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-TGC-ATC-CCC-CAG-GCC-ACC-AT-3′ (SEQ IDNO:3). ISIS 3082 is targeted to the 3′-untranslated region (3′-UTR) ofthe murine ICAM-1 gene. ISIS 3082 is described in Stepkowski et al. (J.Immunol., 1994, 153, 5336).

ISIS 2503 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-TCC-GTC-ATC-GCT-CCT-CAG-GG-3′ (SEQ IDNO:4). ISIS 2503 is targeted to the translation initiation codon of thehuman oncogene, Ha-ras. ISIS 2503 is described in U.S. Pat. No.5,576,208, hereby incorporated by reference.

ISIS 1939 (SEQ ID NO: 2), a phosphorothioate oligonucleotide targeted toa sequence in the 3-untranslated region of ICAM-1 mRNA has been found toexhibit significant biological activity. ISIS 2302 (SEQ ID NO: 1), whichhybridizes to the ICAM-1 mRNA at a position 143 bases 3′ to the ISIS1939 target site was also found to be of similar activity in biologicalassays. Examination of the predicted RNA secondary structure of thehuman ICAM-1 mRNA 3′-untranslated region (Zuker, Science, 1989, 244, 48)surprisingly suggested that both ISIS 1939 and ISIS 2302 hybridize tosequences predicted to be in a stable stem-loop structure of the mRNA.Current dogma suggests that when designing antisense oligonucleotidesregions of RNA secondary structure should be avoided. Thus, ISIS 1939and ISIS 2302 would not have been predicted to inhibit ICAM-1expression.

ISIS 2302 has been found to inhibit ICAM-1 expression in human umbilicalvein cells, human lung carcinoma cells (A549), human epidermal carcinomacells (A431), and human keratinocytes. ISIS 2302 has also demonstratedspecificity for its target ICAM-1 over other potential nucleic acidtargets such as HLA-α and HLA-β. Both ISIS 2302 (SEQ ID NO:1) and ISIS1939 (SEQ ID NO:2) markedly reduced ICAM-1 expression, as detected bynorthern blot analysis to determine mRNA levels, in C8161 human melanomacells. In an experimental metastasis assay, ISIS 2302 decreased themetastatic potential of C8161 cells, and eliminated the enhancedmetastatic ability of C8161 cells resulting from TNF-α treatment. ISIS2302 has also shown significant biological activity in animal models ofinflammatory disease. The data from animal testing has revealed stronganti-inflammatory effects of ISIS 2302 in a number of inflammatorydiseases including Crohn's disease, rheumatoid arthritis, psoriasis,ulcerative colitis, and kidney transplant rejection. When tested onhumans, ISIS 2302 has shown good safety and activity against Crohn'sdisease. Further ISIS 2302 has demonstrated a statistically significantsteroid-sparing effect on treated subjects such that even after fivemonths post-treatment subjects have remained weaned from steroids and indisease remission. This is a surprising and significant findingregarding ISIS 2302's therapeutic effects.

Example 2 Sources of Compounds

In general, the compounds used in the studies described herein areavailable from a variety of commercial sources, or can be synthesizedfrom available reagents by those skilled in the art using methods knownin the art. For sake of convenience, some specific commercial suppliersof the more significant compounds used in, or identified by, the studiesdescribed herein are provided in the following list.

Chol (cholesterol) is purchased from Avanti Polar Lipids, Inc.(Alabaster, Ala.) or from Sigma Chemical Corp. (St. Louis, Mo.).

1-Dodecyl-2-pyrrolidinone is purchased from Aldrich Chemical Co.(Milwaukee, Wis.).

DOPE (dioleoylphosphatidylethanolamine) is purchased from Avanti.

DMPC (dimyristoylphosphatidylcholine) is purchased from Avanti or Sigma.

DPPC (dipalmitoylphosphatidylcholine) is purchased from Sigma, Avanti orGenzyme Corp. (Cambridge, Mass.).

DMPG (dimyristoylphosphatidylglycol) is purchased from Avanti or Sigma.

DMSO (dimethyl sulfoxide) is purchased from Sigma or Aldrich.

IPM (isopropyl myristate, a.k.a. myristic acid isopropyl ester) ispurchased from Sigma or Aldrich.

Menthone is purchased from Aldrich.

1-Methyl-2-pyrrolidinone is purchased from Sigma.

Oleic acid is purchased from Sigma.

PG (propylene glycol, a.k.a. 1,2-propanediol) is purchased from Sigma orAldrich.

Tween 40 [polyoxyethylene(2)sorbitan monopalmitate] is purchased fromSigma or Aldrich.

Azone (dodecyl azone, a.k.a. laurocapram) is purchased from ShanghaiDaniel Chem Technologies Co., Ltd., Shanghai, People's Republic ofChina.

Limonene (d-limonene) is purchased from Sigma.

MIGLYOL™ 818 is purchased from Hhls AG, Marl, Germany.

Example 3 In Vitro Skin Testing

Male and female hairless SKH1 mice 6-8 weeks old were obtained fromCharles River Laboratories (Wilmington, Mass.) and were euthanized usingcarbon dioxide asphyxiation. Fresh and frozen skins were mounted on avertical Franz diffusion cell (Permegear, N.J.) with each skin having adiffusional area of 0.636 cm². Receptor chambers having a volume of 5.1ml were filled with isotonic phosphate buffer (pH 7.2) containing 0.1%(v/v) of 36% aqueous formaldehyde as preservative. Receptor temperatureswere maintained at 37±0.5° C. and stirred continuously at 600 rpm. Theskins were allowed to hydrate for 1 hour prior to starting anexperiment. Experiments generally were performed at 24 hours.

Penetration enhancers/vehicles were added into the donor compartment for1 hour and then washed off with 500 μl of methanol. The total amount ofenhancer/vehicle that was added to each donor compartment was 10 μl(unless otherwise noted). After methanol wash, the skin was lightlywiped and blown dry to remove any visible moisture. In an experimentstudying the effect of methanol on penetration enhancement, no wash wasperformed. Also, in experiments studying the effects of pretreatmenttime, the amount of time the enhancer was allowed to stay on the skinwas varied (i.e., minutes or 1, 2 or 3 hours).

Olignucloetide [i.e., ISIS 2302 (SEQ ID NO:1)] was added on top of theenhancer solution. ISIS 2302 was added to each donor compartment as a200 μl normal saline solution containing both 1 mg of unlabeledoligonucleotide radiolabeled oligonucleotide and approximately 300,000decays per minute (“DPM”) of radiolabeled oligonucleotide. Epidermal,dermal and receptor penetration values are expressed as the ratio of thecounts penetrated versus the control counts.

The following chemicals were used as enhancers/vehicles: propyleneglycol (PG), dimethyl sulfoxide (DMSO), isopropyl myristate (IPM),Azone, MIGLYOL™ 818, oleic acid, d-limonene, limonene,1-dodecyl-2-pyrrolidinone (1dodecyl2pyrrol), 1-methyl-2-pyrrolidinone(1Methyl2pyrrol), menthone, ethanol and TWEEN 40.

Statisical analyses were performed on Excel using Students t-test(two-sample assuming equal variances) along with averages, standarddeviations, and standard errors. Female hairless mice werepreferentially used as the studies progressed due to an uncharacterizedbut recurring follicular infection that appeared to preferentiallytarget male mice.

As shown in FIG. 1, the best epidermal penetration enhancers for thedelivery of ISIS 2302 are isopropyl myristate (“IPM”; 1.67%, 2.14% and3.11%), menthone (2.93%), ethylene glycol (2.41%),1-methyl-2-pyrrolidinone (“1Methyl2pyrrol”; 2.41%), d-limonene (1.55%),MIGLYOL 818®

(1.62%) and dimethyl sulfoxide (DMSO; 1.56%). In contrast, for dermalpenetration, the best penetration enhancers are Tween 40 (1.42%), oleicacid (˜1.0%), d-limonene (0.72%), 1-dodecyl-2-pyrrolidinone(“1dodecyl2pyrrol”; 0.67%), DMSO (0.38%) and 1-methyl-2-pyrrolidinone(“1Methyl2pyrrol”; 0.25%). There is no little or no correlation betweenepidermal penetration enhancement and dermal penetration enhancement, aneffect which may be due to different mechanisms of action of delivery tothe two layers, rates of penetration, the duration of the experiments,the duration of enhancer pretreatments, or a combination of suchfactors.

“Receptor penetration” in the tables refers to the percentage dose thatmigrates through the isolated skin and thus deposits in a receptacle atthe end of the experimental set-up. A high value in this columnindicates the formulation has potential as a systemic delivery vehicle.

Experiments with Azone were carried out to examine how much of a factormethanol is in the delivery of Isis 2302; these results are also shownin FIG. 1. Azone pretreatment with a methanol wash resulted in epidermaland dermal penetration values of 1.31% and 0.16%, respectively, whereasthe values for experiments without methanol values were 0.72% and 0.13%for epidermal and dermal penetration, respectively. Ethanol had littleeffect on the penetration of ISIS 2302 when limonene was used as anenhancer. Higher volumes of limonene and isopropyl myristate did notresult in an increase in the penetration.

Example 4 Cream Formulations and Effects of Oligonucleotide Chemistries

Studies were carried out to optimize the formulation containingisopropyl myristate, and the results are shown in FIG. 2. Duration ofpretreatment ranging from 30 minutes to 3 hours had little effect on thepenetration of ISIS 2302. Lower concentration of isopropyl myristate inthe range of 10 to 35% v/v in water reduced the penetrationsignificantly; however, the coarse mixture of isopropyl myristate andwater applied in very small quantities (10-30 μL) may have resulted inspotty coverage of the skin. Lower amounts of ISIS 2302 resulted in anincrease in the percent of applied dose penetrated.

In order to formulate a cream from isopropyl myristate, its viscositywas increased using oil soluble agents and surfactants such as glycerylmonosterate, stearic acid and bees wax. Oligonucleotide was dissolved ina water phase consisting of aqueous surfactants and viscosity impartingagents such as polyoxyl-40 stearate and polyethylene glycol derivatives.Cream formulations consisting of Water (36-45% w/w), Isopropyl Myristate(30-48% w/w), Glyceryl monostearate (10-16% w/w), Polyoxyl-40 Stearate(0-15% w/w) and antimicrobial preservatives (benzyl alcohol,methylparaben, propylparaben) were studied in vitro for penetration.Oligonucleotide was thoroughly mixed with the cream formulations to givea final concentration of 1 mg oligonucleotide for each 149 mg cream.Appropriate controls were used to determine the radioactivity per mg ofcream.

The cream formulation with 30% isopropyl myristate resulted in anepidermal penetration of 1.66% and a dermal penetration of 1.57% forISIS 2302 (FIG. 2). Similar penetration values were seen with creamformulation containing 48% isopropyl myristate.

A cream formulation of ISIS 15839, a 5-methylcytosine-comprising2′-methoxyethoxy isosequence hemimer derivative of ISIS 2302, with 30%isopropyl myristate showed a very high dermal penetration, i.e., 11% ofthe applied dose. The results presented in FIG. 2 thus demonstrate thatoligonucleotides of different chemical compositions penetrate the skinwhen formulated in isopropyl myristate cream formulations.

Example 5 In Vivo Testing of ICAN-1 Suppression

The oligonucleotide ISIS 3082 (SEQ ID NO:3), which is targeted to themurine ICAM-1 gene, was mixed with empty (“f”) liposomes or encapsulatedinto (“e”) liposomes as set forth below to determine the degree ofICAM-1 suppression effected thereby:

-   -   1. DOPE-f Liposomes (DOPE:DPPC:Chol; 20:60:20% w/w) mixed with        ISIS 3082 to obtain 10 mg/mL ISIS 3082;    -   2. ISIS 3082 solution at 10 mg/mL;    -   3. DOPE-f Liposomes (DOPE:DPPC:Chol; 20:60:20% w/w) mixed with        ISIS 3082 to obtain 10 mg/mL ISIS 3082;    -   4. DOPE-e Liposomes (DOPE:DPPC:Chol; 20:60:20% w/w) with ISIS        3082 encapsulated in the liposomes, not purified, to obtain 10        mg/mL ISIS 3082;    -   5. DMPG-f Liposomes (DMPG:DPPC:Chol; 20:60:20% w/w) mixed with        ISIS 3082 to obtain 10 mg/mL ISIS 3082;    -   6. DMPG-e Liposomes (DMPG:DPPC:Chol; 20:60:20% w/w) with ISIS        3082 encapsulated in the liposomes, not purified, to obtain 10        mg/mL-ISIS 3082;    -   7. DMPC-f Liposomes (DMPC:DPPC:Chol; 20:60:20% w/w) mixed with        ISIS 3082 to obtain 10 mg/mL ISIS 3082;    -   8. DMPC-e Liposomes (DMPC:DPPC:Chol; 20:60:20% w/w) with ISIS        3082 encapsulated in the DMPC liposomes, not purified, to obtain        10 mg/mL ISIS 3082;    -   12. No treatment, phorbol myristate acetate (“PMA”) positive        control; and    -   13. No treatment, no PMA control (“basal”).

Liposome Preparation: The liposomes were prepared by hydrating a dryfilm of lipids in a glass container with either phosphate bufferedsaline at pH 7.4 or a 10 mg/mL solution of ISIS 3082 in PBS. Thehydrated lipids were then extruded 21 times through a 50 nm membrane toform small liposomes with final lipid concentration of ˜100 mg/mL, drugconcentration of ˜10 mg/mL and particle size of ˜100 nm.

Animal Studies: Liposome formulations were applied to the back ofhairless mice using a Hilltop™ chamber (Hilltop Research, Cincinnati,Ohio) that keeps the formulation in place. Three mice were tested ineach group. Forty-eight hours after the formulation application, thetreated part of the skin was challenged with PMA to induce ICAM-1. Micewere sacrificed 4 hours after PMA application and skin collected forNorthern analyses of the mRNA levels, which were performed essentiallyaccording to the protocol detailed in Examples 3 and 7 of co-pendingU.S. patent application Ser. No. 09/062,416, filed Apr. 17, 1998, herebyincorporated by reference.

The results with ISIS 3082 mixed with empty liposomes are as follows:

Relative mRNA Level Formulation (PMA = 100%) Basal 12.46 ± 2.39  DOPE-f(#1) 71.80 ± 7.93  DOPE-f (#2) 64.02 ± 11.32 DMPG-f 63.84 ± 11.54 DPPC-f91.80 ± 0.25  PBS 93.91 ± 11.04

The DOPE and DMPG liposomes show about 30% to about 40% reduction inPMA-induced ICAM-1 expression, whereas the phosphate buffered salinesolution formulation and DPPC liposomes show much lower reduction. Theresults prove that ISIS 3082 penetrates the skin when mixed withliposomes and that the penetration of drug thus achieved is sufficientto cause a biological effect.

The results with ISIS 3082 encapsulated in the liposomes are as follows:

Relative mRNA Level Formulation (PMA = 100%) Basal 12.46 ± 2.39  DOPE-e69.95 ± 5.19  DPPC-e 67.19 ± 11.99 DMPG-e 58.54 ± 12.40The liposome formulations comprising DOPE, DPPC or DMPG andencapsulating ISIS 3082 all show a 30-50% reduction in ICAM-1 mRNA,showing that ISIS 3082 penetrates the skin when encapsulated inliposomes and that the penetration of drug thus achieved is sufficientto cause a biological effect.

Example 6 Comparison of Topical and Systemic Administration ofOligonucleotides

In order to develop a formulation for the local delivery ofoligonucleotides via topical administration, the following experimentswere carried out.

Formulations: A cream formulation of 2% ISIS 2503 (SEQ ID NO:4),intended for topical application, was compared to 20 mg/mL formulationsin saline administered via intravenous, subcutaneous or intradermalmeans.

The cream formulation was prepared by heating the oil phase [containingisopropyl myristate (30% w/w) and glyceryl monostearate (10% w/w)] andthe aqueous phase [containing water (45% w/w) and polyoxyl-40-stearate(15% w/w)] to 70° C. followed by homogenization at 7,000 rpm using aSilverson L4RT mixer (Silverson Machines, East Long Meadows, Mass.),after which the mixture was allowed to cool to room temperature. Theparticle size of the oil phase droplet in the cream had a mean diameterof 1.0 um. ISIS 2503 was mixed with the cream by trituration.

Animal Studies: SCID mice (Taconic Farms, Inc., Germantown, N.Y.) ˜6weeks old, were transplanted with human skin and allowed to establishthe xenograft for 6 weeks. 200 mg cream or 20 mg/kg solution wereadministered at 48, 24 and 4 hours prior to TNF-a administration. TNF-αwas injected in to the xenograft to induce inflammation. Mice weresacrificed and skin isolated for immunohistochemistry.

Stained tissue samples show a pronounced accumulation of theoligonucleotides in the nuclei of the cells in the viable epidermis upontreatment with the cream formulations. Accumulation is also seen in thedermis but no nuclear accumulation is visible. The cream formulationthus provides for the selective delivery of oligonucleotides to cells ofthe dermis.

In contrast, photomicrographs of skin treated intravenously with thesolution formulation show accumulation of oligonucleotide in the dermisbut no nuclear accumulation is visible. There was no accumulation in theepidermis.

Similarly, photomicrographs of skin treated intradermally with thesolution formulation show a large amount of oligonucleotide in theproximity of the needle tract in the dermis and some in the epidermis.Again, however, there is no nuclear accumulation.

Taken together, the preceding results show that oligonucleotidedelivered to the dermis by systemic or direct injection route does notaccumulate in the cells of viable epidermis whereas topical deliverywith the cream formulation can target the viable epidermis. The creamformulation can thus be used to prepare pharmaceutical and otherformulations comprising any of a variety of oligonucleotides, includingbut not limited to those described herein, intended for dermal delivery.The invention thus provides methods for preventing or treating a varietyof dermal disease and disorders, such methods comprising contacting theskin of an animal with a pharmaceutical composition comprising anoligonucleotide according to the present disclosure.

Example 7 IPM Cream Formulations

An oil phase was prepared by dissolving methylparaben (3 mg),propylparaben (4.8 mg), phenoxyethanol (10 mg) and glycerol monostearate(100 mg) in heated isopropyl myristate IPM (100 mg). The aqueous phasewas prepared by dissolving monobasic sodium phosphate monohydrate (3 mg)and dibasic sodium phosphate heptahydrate (9 mg) in a target weight ofwater for a 1 g total formulation. The pH of the aqueous solution wasadjusted to 7±0.2 with 1N monobasic sodium phosphate and 1N sodiumhydroxide. The solution was heated and methylparaben (2 mg),propylparaben (0.2 mg), phenoxyethanol (15 mg) and polyoxyl 40 stearate(150 mg) were then added followed by hydroxypropyl methylcellulose (5mg) and oligonucleotide ISIS-2302 (0.1 mg, 1 mg, 5 mg and 20 mg). Theoil phase was then added to the water phase while homogenizing to formthe emulsion which was then cooled to room temperature.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

1. A pharmaceutical composition comprising an oligonucleotide admixedwith a topical delivery agent comprising a penetration enhancing amountof isopropyl myristate, wherein said composition is formulated into acream or emulsion, with the proviso that said oligonucleotide is not anoligonucleotide that is complementary to a portion of a mRNA sequencecoding for tumor necrosis factor and inhibits the expression of thetumor necrosis factor.
 2. The composition of claim 1, wherein saidcomposition comprises 10% to 50% isopropyl myristate.
 3. The compositionof claim 1, wherein said composition comprises about 30%-48% isopropylmyristate.
 4. The composition of claim 1, wherein said compositionfurther comprises 10%-16% glyceryl monostearate.
 5. The composition ofclaim 1, wherein said composition further comprises glyceryl monooleate.6. The composition of claim 1, wherein said composition furthercomprises up to 15% polyoxl-40-stearate.
 7. The composition of claim 1,wherein said composition further comprises polyoxylethylene-20-cetylether.
 8. The composition of claim 1, wherein said composition furthercomprises glyceryl monostearate.
 9. The composition of claim 1, whereinsaid composition further comprises 5% to 20% glyceryl monostearate. 10.The composition of claim 1, wherein said composition comprises about 2%of said oligonucleotide.
 11. The composition of claim 1, wherein saidcomposition is formulated into a cream.
 12. The composition of claim 1,wherein said composition is formulated into an emulsion.
 13. Thecomposition of claim 1, wherein said oligonucleotide comprises at leastone modified internucleoside linkage, at lease one nucleoside comprisinga modified sugar, or at least one nucleobase comprises a modifiednucleobase.
 14. The composition of claim 13, wherein eachinternucleoside linkage is a phosphorothioate internucleoside linkage.15. The composition of claim 13, wherein at least one modified sugarcomprises a 2′-O-methoxyethyl.
 16. The composition of claim 13, whereinthe modified nucleobase is a 5-methylcytosine.
 17. The composition ofclaim 1, wherein the oligonucleotide is about 8 to about 100 nucleotidesin length.
 18. The composition of claim 1, wherein the oligonucleotideis about 10 to about 30 nucleotides in length.
 19. The composition ofclaim 1, wherein the oligonucleotide consists of 20 linked nucleosides.20. The composition of claim 1, wherein the oligonucleotide is asingle-stranded oligonucleotide.
 21. The composition of claim 1, whereinsaid oligonucleotide is a gapmer.
 22. The composition of claim 1,wherein said oligonucleotide is about 10 to 30 nucleotides in length,wherein said oligonucleotide is a gapmer having a centraldeoxynucleotide region flanked on each of the 5′ and 3′ ends of saiddeoxynucleotide region with a wing region comprising at least one2′-O-methoxyethyl substituted nucleoside, wherein each internucleosidelinkage in said oligonucleotide is a phosphorothioate linkage, andwherein each cytosine in said oligonucleotide is a 5-methylcytosine. 23.The composition of claim 1, wherein the oligonucleotide is apharmaceutically acceptable salt.
 24. A pharmaceutical composition fortopical delivery of an antisense oligonucleotide, comprising: anantisense oligonucleotide; a penetration enhancing amount of isopropylmyristate; and 10%-16% glyceryl monosterate, wherein said composition isformulated into a cream or emulsion, with the proviso that saidantisense oligonucleotide is not an oligonucleotide that iscomplementary to a portion of a mRNA sequence coding fox tumor necrosisfactor and inhibits the expression of the tumor necrosis factor.
 25. Thecomposition of claim 24, wherein said composition comprises 10% to 50%isopropyl myristate.
 26. The composition of claim 24, wherein saidcomposition comprises about 30%-48% isopropyl myristate.
 27. Thecomposition of claim 24, wherein said composition further comprises upto 15% polyoxl-40-stearate.
 28. The composition of claim 24, whereinsaid composition comprises about 2% of said antisense oligonucleotide.29. The composition of claim 24, wherein said composition is formulatedinto a cream.
 30. The composition of claim 24, wherein said compositionis formulated into an emulsion.
 31. The composition of claim 24, whereinsaid antisense oligonucleotide comprises at least one modifiedinternucleoside linkage, at lease one nucleoside comprising a modifiedsugar, or at least one nucleobase comprising a modified nucleobase. 32.The composition of claim 31, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.
 33. The composition of claim31, wherein at least one modified sugar comprises a 2′-O-methoxyethyl.34. The composition of claim 31, wherein the modified nucleobase is a5-methylcytosine.
 35. The composition of claim 24, wherein the antisenseoligonucleotide is about 8 to about 100 nucleotides in length.
 36. Thecomposition of claim 24, wherein the antisense oligonucleotide is about10 to about 30 nucleotides in length.
 37. The composition of claim 24,wherein the antisense oligonucleotide consists of 20 linked nucleosides.38. The composition of claim 24, wherein the antisense oligonucleotideis a single-stranded oligonucleotide.
 39. The composition of claim 24,wherein said antisense oligonucleotide is a gapmer.
 40. The compositionof claim 24, wherein said antisense oligonucleotide is about 10 to 30nucleotides in length, wherein said antisense oligonucleotide is agapmer having a central deoxynucleotides region flanked on each of the5′ and 3′ ends of said deoxynucleotides region with a wing regioncomprising at least one 2′-O-methoxyethyl substituted nucleoside,wherein each internucleoside linkage in said antisense oligonucleotideis a phosphorothioate linkage, and wherein each cytosine in saidantisense oligonucleotide is a 5-methylcytosine.
 41. The composition ofclaim 24, wherein the antisense oligonucleotide is a pharmaceuticallyacceptable salt.
 42. The composition of claim 22, wherein saidcomposition comprises 10% to 50% isopropyl myristate.
 43. Thecomposition of claim 40, wherein said composition comprises 10% to 50%isopropyl myristate.